A Study on the Activators and Targets of NF-κB in Human Cancer Cells

The ubiquitous transcription factor, NF-κB, is deregulated in many solid and hematological malignancies. Most of the tumor types and cell lines show constitutive activation of NF-κB that enables them to resist chemo and/or radio therapy by inducing the expression of anti apoptotic/survival factors. In addition NF-κB enhances the ability of tumors to metastasize as well as form new vasculature, which are critical for tumor survival. Since constitutive NF-κB activity correlates positively to the resistance of cancer cells to anti cancer agents, antagonizing NF-κB offers an immense therapeutic potential to improve the clinical response in these tumor types. Many studies have shown that downregulation of NF-κB sensitizes cells to therapy as well as inhibits the metastatic and angiogenic ability of different tumor types. Recently we have reported that NF-κB is constitutively activated in high grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix. However, the mechanisms by which tumors maintain a sustained activation of NF-κB are not clearly defined. Since ex vivo tissue explants are not very much suited for mechanistic studies, the present proposal is aimed at identifying the factors responsible for the activation of NF-κB and their targets in human cervical cancers using in vitro model systems. Hence it is proposed to study the expression of activators of NF-κB and their targets which are often responsible for tumor initiation, progression, invasion and metastasis. It is proposed to test the hypothesis that cytokines and growth factors secreted by tumor cells as well as the surrounding stromal layer are responsible for constitutive activation of NF-κB. Organotypic raft culture will be used as a mechanistic model to test this hypothesis and it will also serve as an in vitro model for testing the efficacy of anti cancer drugs.

A study on the transforming growth factor-β signaling intermediate, Smad3, in human cervical cancer

Cancer cells may lose their sensitivity and responsiveness to TGF-β due to loss of TGF-β or functional components of the TGF-β signaling pathway promoting the development of cancer. Defective TGF-β signaling due to mutations in Smads has been reported in several human cancers. Recently our laboratory identified loss of expression and mutations in Smads 2 and 4. In the present study human cervical cancer cells and tumor samples will be analyzed for variations in the expression of Smad 3 and/or its mutations. Semi-quantitative RT PCR and Western blotting will be used to assess the changes in expression at the RNA and protein levels, respectively. PCR-SSCP-sequencing or RTPCR-SSCP-sequencing approaches using isolated DNA and RNA, respectively, will be used for the mutation analysis. Successful completion will enhance our understanding on the role of Smads in general and that of Smad 3 in particular in human cervical cancer.

Functional Characterization of miRNAs in Human Cancer Cells

Cancer will be one of the major diseases in India by 2025. Initiation, promotion, malignant conversion, progression and metastasis are the five major steps in cancer development. Combined interaction of both tumor suppressors and inducers is the cause for the formation of cancer. Even though several genes had been identified in human, the mechanism of cancer formation is yet to be ascertained. A recently identified class of non-protein coding small RNAs, micro RNAs (miRNA), may provide new insight in cancer research. miRNAs are short, non-coding RNAs that regulate gene expression by controlling the efficiency of mRNA translation. Many miRNAs exhibit altered expression levels in cancer, and scientific community is only starting to understand the functional consequences of the loss or gain of particular miRNAs to cancerous phenotype. miRNAs are also naturally occurring triggers of the RNA-interference pathway. miRNAs are expected to regulate more than 30% of all mRNAs post-transcriptionally.
Several groups have studied the miRNA expression in cancer patients and found that miRNAs are differentially expressed in normal and cancer tissues. Some miRNAs may function as oncogenes or tumor suppressor genes. Examples of their roles in development and disease are rapidly accumulating but the biological functions of most of them are completely unknown. Here we propose to elucidate the functions of reported miRNAs in cancer cells in the following manner. 1. Over-expression of miRNAs 2. Knocking down miRNAs using genetic approaches 3. Silencing the miRNAs using antagomirs (single strand RNA) and siRNAs 4. Replacement of miRNAs (in case of tumor suppressor miRNAs). The outcome of these results will be compared with computational predictions of the target.  Up- and/or down-regulated expression of the candidate miRNAs are a good approach to study the function of miRNAs in cancer pathogenesis. It will also be important to elucidate biochemical pathways that affect miRNA function as well as environmental and genetic factors that affect miRNA expression. Once this information is available, a rational approach can be taken to design new therapeutic strategies that aim to treat cancer.

Studies on TGF-b-mediated changes in gene expression and signaling pathways in human cervical cancer cells

Transforming growth factor-β (TGF-β) family includes a large number of structurally related polypeptide growth factors-TGF-βs, activins and bone morphogenetic proteins-that are important in homeostasis and repair of virtually all tissues. TGF-βs were identified as regulators of mesenchymal growth and differentiation, cell cycle arrest in epithelial and hematopoetic cells, extracellular matrix production and immunosuppression. We assessed the responsiveness of six human cervical cancer cell lines to TGF-β with p3TP-lux reporter assay and found that HeLa and SiHa cells were highly responsive to TGF-β. However, when pSBE4-BV/Luc reporter having 4 smad binding elements was used, only the SiHa but not HeLa cells showed Smad activation. Smad DNA binding activity was relatively more in SiHa than HeLa cells upon TGF-β treatment and the active complex contained Smad 2 and Smad 4. In MTT assays, HeLa cells treated with 5ng/ml of TGF-β for 24h showed proliferation whereas SiHa cells showed growth inhibition under the same conditions. TGF-β treatment resulted in G0/G1 arrest with a reduction in S-phase only in SiHa cells and a chemical inhibitor of Smad activation (SB203580) blocked the growth inhibitory effect of TGF-β in SiHa while the proliferative response in HeLa was unaffected. TGF-β-induced translocation of phospho-Smad 2 was relatively less in HeLa than SiHa cells. MAPK activation occurred within 5 min and persisted up to 15 min upon TGF-b treatment in HeLa but was negligible in SiHa cells. TGF-β activated JNK in HeLa but SiHa cells showed a down-regulation of its activity. When an inhibitor of MAPK (U0126) was used, the TGF-β-mediated proliferative response in HeLa cells was completely abolished. SB203580 did not affect MAPK activation induced by TGF-β in HeLa cells. We reported for the first time an activation of TGF-β -dependent MAPK within 5 min of treatment that resulted in cell cycle progression in a cervical adenocarcinoma cell line, HeLa, which is presumably independent of Smad activation. Hence, in the present proposal it is planned to study smad independent mechanisms by down regulation of smads by SiRNA or antisense approaches. Studies on TGF-β-mediated changes in MAPK, Rho, Rac and Rock signaling pathways in human cervical cancer cells will also be undertaken. Micro array approaches to study differential regulation in cervical cancer cells are also planned.

Studies on the mechanisms of apoptosis induced by organosulfur compounds from garlic                                               

Garlic (Allium sativum), a member of the Allium family, is one of the earliest documented plants for its varied uses and texts of ancient times endorsed its usage in medicine and also for religious purposes (Rivlin, 2001). Egyptians, Greeks, Chinese and Indians used garlic for centuries for treating various ailments such as heart disease, arthritis, pulmonary complaints, abdominal growths (particularly uterine), diarrhea, and worm infestation (Rivlin, 2001; Rivlin et al., 2006). Extensive research on garlic points to the fact that major biological effects of garlic can be attributed to the innumerable chemical compounds present within garlic (Sparnins et al., 1988; Wargovich et al., 1988). Garlic is rich in organosulfur compounds and more than 33 different organosulfur compounds are present within garlic. Many of the organosulfur compounds, the major active principles in garlic, inhibited the proliferation of cancer cells and some of them induced apoptosis in tumor cells of different tissue origin (Dirsch et al., 1998; Kwon et al., 2002; Pinto et al., 2001; Shirin et al., 2001; Sigounas et al., 1997). Allicin is one of the major organosulfur compounds produced in freshly crushed garlic and is responsible for its characteristic odour. It is biologically very active but is highly unstable and decomposes into mostly diallyl disulfide (DADS), diallyl trisulfide (DATS), ajoene, S-allyl mercapto cysteine, S-allyl cysteine and vinyl dithiines (Dirsch et al., 1998; Hirsch et al., 2000; Sigounas et al., 1997; Sundaram and Milner, 1993; Welch et al., 1992). DATS is reported to be having a potent antiproliferative activity in comparison to diallyl disulfide and diallyl sulfide in A549 cells. It was found to induce apoptosis in A549 cells. Hence, apoptosis could be a potential general mechanism providing a mechanistic basis for the anticarcinogenic activities of DATS (Sakamoto et al., 1997). Some of the garlic compounds have antioxidant properties that may lead to the inhibition of the transcription factor nuclear factor- kappa B (NF-κB). Earlier reports by other researchers show that compounds from garlic may activate or inhibit NF-κB, a transcription factor that is known to protect cells from apoptosis induced by anticancer agents. Interestingly inhibitors of NF-κB such as curcumin enhanced the apoptosis inducing effects of anticancer agents such as taxol thereby showing a synergistic effect (Bava et al., 2005). Garlic powder extracts altered cytokine levels in human blood sample supernatants reduced NF-κB activity in human cells exposed to these samples (Keiss et al., 2003). S-allyl cysteine was found to inhibit the activation of NF-κB in human T cells (Geng et al., 1997). Ajoene was demonstrated to activate nuclear translocation of the transcription factor NF-κB in HL-60 leukemic cells (Dirsch et al., 1998).Our research group has recently reported that allicin inhibits the growth of cancer cells of murine and human origin (Oommen et al., 2004). Allicin induced the formation of apoptotic bodies, nuclear condensation and a typical DNA ladder in cancer cells. Furthermore, activation of caspases-3, -8 and -9 and cleavage of poly (ADP-ribose) polymerase were induced by allicin (Oommen et al., 2004). These results demonstrating allicin-induced apoptosis of cancer cells are novel since allicin has not been shown to induce apoptosis previously. We continued this work essentially focusing on the metabolites of allicin such as DAS, DADS and DATS. Our preliminary data suggest that DATS can efficiently induce apoptosis of cancer cells and is a more effective inducer of apoptosis than DADS and DAS. However, the mechanism of apoptosis is not clear at present. The present proposal aims to study the mechanism of apoptosis induced by DATS and that of organosulfur compounds derived from garlic other than allicin such as ajoene.